PDF电子书：Current Protocols in Cell Biology 2010版

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- T* D* X9 k' n! ~Current Protocols in Cell Biology 2010年完整版 5483页
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3 q/ P, C  a8 Y3 uOnline ISBN: 9780471143031  Y7 Q; R* P( J# I7 a4 R( j
DOI: 10.1002/0471143030) C, U8 i4 t% s: f; R, O6 ]/ N
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Table of Contents
6 a$ z( @6 N' R9 B" J/ e3 f1. Preface
' u5 x5 |& v0 M6 p5 k! ?2. Foreword
2 }, y. P; S+ C/ B( a4 Y4 K! A2 u3. Chapter 1 Cell Culture
; I/ ~. K( f) C3 t: f- p1. Introduction1 ], u6 k& _8 p( W- W- A: ]
2. Unit 1.1 Basic Techniques in Mammalian Cell Tissue Culture4 P# n( ]0 @6 `) m" @5 h5 N2 S* e
3. Unit 1.2 Media for Culture of Mammalian Cells6 D. _) f! |/ q1 `" w0 x) k
4. Unit 1.3 Aseptic Technique for Cell Culture
2 P9 ^! A5 f8 S5. Unit 1.4 Sterilization and Filtration4 W9 H3 q8 \8 r: A, W
6. Unit 1.5 Assessing and Controlling Microbial Contamination in Cell Cultures
& p, f! q; F4 [$ V1 v' B% @4 r7. Unit 1.6 Media and Culture of Yeast/ \. d7 L5 \9 K* |
8. Unit 1.7 BY-2 Cells: Culture and Transformation for Live Cell Imaging
5 N' [) p9 K& @( C: N4. Chapter 2 Preparation and Isolation of Cells" @; K! t1 Q' X0 k3 ^* o$ P# H
1. Introduction
4 K. e& I( T& r0 M# J0 z2. Unit 2.1 Establishment of Fibroblast Cultures& h) J- a% w( ]7 Z
3. Unit 2.2 Preparation and Culture of Human Lymphocytes
, L6 f% H9 t+ N9 a4. Unit 2.3 Preparation of Endothelial Cells
* E$ a$ k7 b& Q* u8 }8 O5. Unit 2.4 Generation of Continuously Growing B Cell Lines by Epstein-Barr Virus Transformation
. y% ~6 T  ]2 @3 A3 \; p# V: |6. Unit 2.5 Laser Capture Microdissection- ^1 ]! x# i' T5 q+ \' g* b8 h# Y
7. Unit 2.6 Preparation of Human Epidermal Keratinocyte Cultures
1 x# E4 F& k+ n" e4 i8. Unit 2.7 Preparation and Coculture of Neurons and Glial Cells5 j# \6 D' V3 F  x0 ^* {
5. Chapter 3 Subcellular Fractionation and Isolation of Organelles
. S: u2 @) a' a# r0 l. h1. Introduction6 I7 \  Z; ?5 E9 l% O& @  n4 n
2. Introduction% ~+ ~; e3 O. P. C
3. Unit 3.1 Overview of Cell Fractionation& F  Y3 e" {( Y4 F/ e  X
4. Unit 3.2 Isolation of Rat Hepatocyte Plasma Membrane Sheets and Plasma Membrane Domains
$ M& A8 C2 u; D4 Z1 Q* f( X5. Unit 3.3 Isolation of Mitochondria from Tissues and Cells by Differential Centrifugation
) C2 g, q- W! ^! _- H  i. e8 I6. Unit 3.4 Purification of a Crude Mitochondrial Fraction by Density-Gradient Centrifugation0 F4 x+ X& T5 f) b
7. Unit 3.5 Isolation of Peroxisomes from Tissues and Cells by Differential and Density Gradient' }  f7 j7 q) x$ J
Centrifugation
& v8 e; h) W6 W- N+ U. x8. Unit 3.6 Isolation of Lysosomes from Tissues and Cells by Differential and Density Gradient- b) H# [: d9 W1 u' W* @: X" }
Centrifugation
2 c% T6 q/ q! _( s# z9. Unit 3.7 Overview of Subcellular Fractionation Procedures for the Yeast Saccharomyces cerevisiae4 M- B" L' p7 I/ T' v7 n" L' C$ Y) w- Y
10. Unit 3.8 Isolation of Subcellular Fractions from the Yeast Saccharomyces cerevisiae: A1 c$ G3 }9 r* d/ _4 p; {
11. Unit 3.9 Isolation of Golgi Membranes from Tissues and Cells by Differential and Density Gradient
6 e. O1 k) v1 @Centrifugation
' R* k( C$ n7 m" G0 H& ~* ]+ o12. Unit 3.10 Isolation of Nuclei and Nuclear Membranes From Animal Tissues
& x$ x, `7 U6 o; x6 x13. Unit 3.11 Free-Flow Electrophoretic Analysis of Endosome Subpopulations of Rat Hepatocytes
( N. q; X3 m: M14. Unit 3.12 Isolation of Synaptic Vesicles, M- B: O! V" o/ B$ H: X
15. Unit 3.13 Isolation of Clathrin-Coated Vesicles by Differential and Density Gradient Centrifugation
) s4 L1 H9 U% `/ m& K4 t9 n16. Unit 3.14 Isolation of Melanosomes
+ }' \% I0 ?4 {5 o17. Unit 3.15 Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation
# r1 W2 u' P* S% r1 |18. Unit 3.16 Isolation of Mast Cell Granules
5 J% z% D# z1 Z4 ]$ a) f/ A6 ?2 p2 q19. Unit 3.17 Immunoisolation of Centrosomes from Drosophila melanogaster+ k1 g6 U6 j) i! ]" Y7 |8 E& M! p. U
20. Unit 3.18 Isolation of Zymogen Granules from Rat Pancreas
! a- X" q0 D  i5 R0 R21. Unit 3.19 Isolation of Glyoxysomes from Pumpkin Cotyledons
5 \  j% ~! n( M! J# P6 w2 d22. Unit 3.20 Isolation of GLUT4 Storage Vesicles) h: h. B5 v! q  Q8 Z+ z( h+ S
23. Unit 3.21 Isolation of Intestinal Brush-Border Membranes
9 ^7 e: U5 A" |3 u( v7 i5 I! D24. Unit 3.22 Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological
+ Z  N% O1 F$ N* WFluids
$ s# F2 ~# _/ z% a' q. ]6 d- f8 g5 W25. Unit 3.23 Isolation of Intermediate Filaments
) T* n& m/ m! K+ Y6 _: g1 e" D2 f26. Unit 3.24 Isolation of T-Tubules from Skeletal Muscle" a* M/ ~) F5 X' C  ?. r: C/ W- Z
27. Unit 3.25 Isolation of Myelin" C5 i8 A1 o0 }7 f( {
28. Unit 3.26 Isolation of Renal Brush Borders
9 |, O1 o2 q* r. s- n$ q29. Unit 3.27 Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane
: H/ H( F) K" S4 R( M6 s( j9 FFractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts
8 r* m) Q/ x/ N- t  r9 _30. Unit 3.28 Isolation of Amyloplasts
7 u  b0 H9 K2 ?* N: Y31. Unit 3.29 Isolation of Microtubules and Microtubule Proteins3 c  x; b# |, ?% _* ]
32. Unit 3.30 Purification of Intact Chloroplasts from Arabidopsis and Spinach Leaves by Isopycnic
8 K5 u. l+ J9 ]4 A" nCentrifugation
* K$ ^* r  ~% d  \3 j! b33. Unit 3.31 Isolation of Neuromelanin Granules/ G& Y! j1 r3 B
34. Unit 3.32 Isolation of Dense Core Secretory Vesicles from Pancreatic Endocrine Cells by Differential and
- N8 e* H7 D3 QDensity Gradient Centrifugation
7 O; P- c1 w1 N) h' Q2 A35. Unit 3.33 Isolation and Biochemical Characterization of Amyloid Plaques and Paired Helical Filaments
/ H0 n0 _* X& f& I7 e36. Unit 3.34 Isolation of Legionella-Containing Vacuoles by Immuno-Magnetic Separation* `; q: V/ i+ d9 T8 Q; H
37. Unit 3.35 Isolation of Platelet Granules
! d' [8 w# D/ T$ y38. Unit 3.36 Isolation of Nucleoli
  o1 y% l9 N* [, G39. Unit 3.37 Isolation of Cytotoxic T Cell and NK Granules and Purification of Their Effector Proteins
) d1 ?$ [6 e5 `40. Unit 3.38 Isolation of Aggresomes and Other Large Aggregates
! s+ m! J5 h, I5 h& s. l" O41. Unit 3.39 Isolation of Chromaffin Granules* [* e9 M/ D# U+ ]$ t: W  C
42. Unit 3.40 Purification of Ribosomes from Human Cell Lines) v, d5 u; K& _  j% V! e
6. Chapter 4 Microscopy
3 o: ^) C; g( c7 c7 k" ]1. Introduction" V) {! z- G3 x- k+ W3 |
2. Unit 4.1 Proper Alignment and Adjustment of the Light Microscope9 i) O$ v7 U6 c" H
3. Unit 4.2 Fluorescence Microscopy
( `3 J6 x! [4 T" x* d4 F* e7 t4. Unit 4.3 Immunofluorescence Staining
: s+ s% w, M# b! g; {! m3 @( _" K5. Unit 4.4 Fluorescent Staining of Subcellular Organelles: ER, Golgi Complex, and Mitochondria$ C# P: |( {  Q
6. Unit 4.5 Basic Confocal Microscopy$ X  Y  A% `8 r) y& P
7. Unit 4.6 Immunoperoxidase Methods for Localization of Antigens in Cultured Cells and Tissues
+ A/ z9 Q) X9 ]5 B8. Unit 4.7 Cryo-Immunogold Electron Microscopy
4 A7 U9 q' [$ J9. Unit 4.8 Correlative Video Light/Electron Microscopy
! {3 K/ H. R0 c10. Unit 4.9 Polarization Microscopy
8 h7 J2 {$ i5 }( r& Z11. Unit 4.10 Fluorescent Speckle Microscopy (FSM) of Microtubules and Actin in Living Cells2 w2 T0 T' c1 v* K5 {
12. Unit 4.11 Two-Photon Excitation Microscopy for the Study of Living Cells and Tissues* G3 \( [. {- A+ U+ L* V. O
13. Unit 4.12 Total Internal Reflection Fluorescence Microscopy for High-Resolution Imaging of Cell-Surface0 }! a" A. P5 @  y$ c
Events
% j0 k  m+ k3 @; @14. Unit 4.13 Fluorescent Labeling of Yeast
: n+ e- ?5 \  L: _, E2 P. o15. Unit 4.14 Fluorescence Lifetime Imaging Microscopy
& _+ b8 j- _! [0 F. E1 e! R16. Unit 4.15 Biological Second and Third Harmonic Generation Microscopy
4 |$ v0 u6 K+ t" {+ ^& F17. Unit 4.16 Analyzing Real-Time Video Microscopy: The Dynamics and Geometry of Vesicles and Tubules
2 X' i8 V" C7 o7 ?in Endocytosis
2 K/ f8 c. m' a7 b4 U: X( e5 C18. Unit 4.17 Scanning Electron Microscopy of Cell Surface Morphology' g5 M2 o3 T* [& n$ R
19. Unit 4.18 Fluorescence Imaging Techniques for Studying Drosophila Embryo Development
3 N" S* u0 e* h20. Unit 4.19 Quantitative Colocalization Analysis of Confocal Fluorescence Microscopy Images
0 r5 n. m0 _: y8 E( x, x% x21. Unit 4.20 Visualizing Protease Activity in Living Cells: From Two Dimensions to Four Dimensions( O) ~2 m4 |: G! R% _+ |7 L
22. Unit 4.21 Photoactivated Localization Microscopy (PALM) of Adhesion Complexes
( `5 A- B( E0 m5 d' k; Y# f23. Unit 4.22 Culturing MDCK Cells in Three Dimensions for Analyzing Intracellular Dynamics
6 N0 }: T" |2 h: y, L* ^24. Unit 4.23 Interference Reflection Microscopy5 y" u$ r7 o, N2 Q/ A
25. Unit 4.24 Fluorescence Correlation Spectroscopy in Living Cells: A Practical Approach
; p6 W4 G5 v) f8 w# I* c" j% R26. Unit 4.25 Analysis of Mitochondrial Dynamics and Functions Using Imaging Approaches
7 r! M) O0 ^5 v0 R% m. G3 u27. Unit 4A Organelle Atlas: Appendix to Chapter 4# ^$ M' o) Q5 }
7. Chapter 5 Characterization of Cellular Proteins
  ]2 S; Z0 s# X8 ^1. Introduction9 k; c) r$ ]3 _$ v6 g; R
2. Unit 5.1 Overview of the Physical State of Proteins Within Cells
7 V) h. N+ F  B+ m: P3. Unit 5.2 Determining the Topology of an Integral Membrane Protein" Q! G( l7 f  _( X- h# w- V5 J
4. Unit 5.3 Determination of Molecular Size by Zonal Sedimentation Analysis on Sucrose Density Gradients) C( z* _% U4 R% J' Z( w
5. Unit 5.4 Analysis of the Association of Proteins with Membranes* f* @6 k' u. t% F7 |/ |
6. Unit 5.5 Determination of Molecular Size by Size-Exclusion Chromatography (Gel Filtration)
1 g9 U; u1 r8 ]4 G7. Unit 5.6 Identification of Proteins in Complex Mixtures Using Liquid Chromatography and Mass
# c2 L- E; U0 p) `8 k1 w" `5 Z, sSpectrometry
* j# Y! T+ ?0 K8. Unit 5.7 Determining Membrane Protein Topologies in Single Cells and High-Throughput Screening
" t3 ~* m7 K, A) n  WApplications( V6 a4 t3 h9 t& t4 [6 P
8. Chapter 6 Electrophoresis and Immunoblotting9 R0 k% P; Q" i) _+ p* L( w% g  u
1. Introduction
1 H6 d. J$ |! ?/ b- ^; y2. Unit 6.1 One-Dimensional SDS Gel Electrophoresis of Proteins
6 I. x1 F- q7 s/ y  n3. Unit 6.2 Immunoblotting and Immunodetection2 C( _) o6 f( B& u; _
4. Unit 6.3 Detection and Quantitation of Radiolabeled Proteins in Gels and Blots" C2 @$ Z$ {  w
5. Unit 6.4 Two-Dimensional Gel Electrophoresis
5 O' O  h8 q5 I' `* o- S' M6. Unit 6.5 One-Dimensional Electrophoresis Using Nondenaturing Conditions" K/ o3 g( n* T6 q
7. Unit 6.6 Staining Proteins in Gels
; E$ d3 u: _/ R8. Unit 6.7 Agarose Gel Electrophoresis of Proteins7 N; Y3 C9 Q. U" h/ O6 }9 w7 \! t
9. Unit 6.8 Fluorescence Detection of Glycoproteins in Gels and on Electroblots
" B/ a2 ~  S/ C) X' }+ m# m% d10. Unit 6.9 Digital Electrophoresis Analysis
" o# {+ B% S" G/ p11. Unit 6.10 Two-Dimensional Blue Native Polyacrylamide Gel Electrophoresis
, p6 v9 [9 L1 ~4 |, L" A% e: \12. Unit 6.11 Measurement of Oxidatively-Induced Clustered DNA Lesions Using a Novel Adaptation of
" W( C- P4 ^, c; [& E! ESingle Cell Gel Electrophoresis (Comet Assay)
7 c  r$ e6 G# L2 y1 T4 _4 P8 m7 L4 h9. Chapter 7 Protein Labeling and Immunoprecipitation- H$ i- A& S. `% ?2 S6 v5 d1 r9 T/ w
1. Introduction' P5 L$ t% G+ `& Y
2. Unit 7.1 Metabolic Labeling with Amino Acids
1 {9 m8 D. F& R5 A3. Unit 7.2 Immunoprecipitation
1 j3 a4 S! B, o! m3 l/ Y4. Unit 7.3 Metabolic Labeling with Sulfate
8 }/ U( |- N6 l4 R: I5 M9 F5. Unit 7.4 Metabolic Labeling with Fatty Acids
) Z' [' y- O$ R9 M8 E6. Unit 7.5 Metabolic Labeling of Prenyl and Carboxyl-Methyl Groups
# c# u7 G3 ^9 p& {+ J/ ]7. Unit 7.6 Metabolic Labeling and Immunoprecipitation of Yeast Proteins
7 F2 A+ O0 ^/ u) G8. Unit 7.7 Metabolic Labeling and Immunoprecipitation of Drosophila Proteins$ c+ w4 Q2 L0 r& A1 O( E" S0 ^
9. Unit 7.8 Metabolic Labeling of Glycoproteins with Radioactive Sugars  p5 ]& {7 t" s
10. Unit 7.9 Analysis of Oxidative Modification of Proteins; B9 l8 ~6 V! Z( G' }$ S+ E' t
11. Unit 7.10 Radioiodination of Cellular Proteins4 J$ @5 |# d8 H. d8 i- |
10. Chapter 8 Cell Cycle Analysis/ W  [+ V1 j- C+ _& c/ W; u
1. Introduction1 ?4 E* k6 O- Y. M5 M
2. Unit 8.1 Overview of the Cell Cycle
, w6 y( _' h+ y4 L3. Unit 8.2 Assays for CDK Activity and DNA Replication in the Cell Cycle
; @  a7 x" d- z3 d0 A4. Unit 8.3 Methods for Synchronizing Cells at Specific Stages of the Cell Cycle
) W- }5 X8 V$ D6 N5. Unit 8.4 Determining Cell Cycle Stages by Flow Cytometry$ ?, L9 T' X. x+ s7 B
6. Unit 8.5 Centrifugal Elutriation to Obtain Synchronous Populations of Cells
1 t5 Z; C: y, z! \7. Unit 8.6 Dynamic Proliferation Assessment in Flow Cytometry
) {" u! F1 m( [5 l8 \$ F: |& p11. Chapter 9 Cell Adhesion
+ P* r) i3 c7 O: ]4 w1. Introduction, d4 E3 ~: h  w) @% n
2. Unit 9.1 Cell-Substrate Adhesion Assays( L, x( U8 y+ D9 R" B: B
3. Unit 9.2 Quantitative Measurement of Cell Adhesion Using Centrifugal Force
3 f# A5 j5 S/ k; }1 }/ k4. Unit 9.3 Cadherin-Dependent Cell-Cell Adhesion/ Z  i4 [6 S9 \& s, C' G4 `
5. Unit 9.4 Analyzing Integrin-Dependent Adhesion- d  I7 A+ M5 A$ p6 k
6. Unit 9.5 Analysis of Cell-Cell Contact Mediated by Ig Superfamily Cell Adhesion Molecules. I( O7 B8 a7 c+ \0 b+ o, C  @
7. Unit 9.6 Measurement of Adhesion Under Flow Conditions
. N$ t2 f5 G8 Z- Q$ [2 Q12. Chapter 10 Extracellular Matrix
# J$ D1 [9 ?7 H  J& z' p* u" _9 B1. Introduction5 M8 d5 F5 g3 a5 @2 f5 k" r" {5 M
2. Unit 10.1 Overview of Extracellular Matrix/ L) N0 l/ z# w  o7 U- L1 _
3. Unit 10.2 Preparation of Basement Membrane Components from EHS Tumors
3 ?9 Q, D1 D7 L4 _+ N4. Unit 10.3 Preparation of Gelled Substrates7 x8 O. c2 t4 _% n) ~6 [3 m8 a
5. Unit 10.4 Preparation of Extracellular Matrices Produced by Cultured Corneal Endothelial and PF-HR9  v/ u5 g5 T: ~/ C, Q
Endodermal Cells/ J8 y; e/ e' D- D* {% h
6. Unit 10.5 Purification of Fibronectin/ t: b( `; L8 M: l8 c! J& l
7. Unit 10.6 Purification of Vitronectin
2 v: J; {) z- L4 ]; e# `# A/ k8. Unit 10.7 Proteoglycan Isolation and Analysis
% D3 D# a. `( \6 N9. Unit 10.8 Matrix Metalloproteinases9 a/ T; a  Z& A3 c. N$ n' [
10. Unit 10.9 Preparation of Extracellular Matrices Produced by Cultured and Primary Fibroblasts1 g( l4 S  r/ T. d0 j( _
11. Unit 10.10 Purification and Analysis of Thrombospondin-1, {; ]7 z$ \0 z* U" F! v
12. Unit 10.11 Purification of SPARC/Osteonectin  M# C7 V5 X' m4 T( [2 n
13. Unit 10.12 Analysis of Fibronectin Matrix Assembly) T' f$ b' a; Q; G2 a0 w
14. Unit 10.13 Non-Radioactive Quantification of Fibronectin Matrix Assembly
$ R( K& j6 f# @( l+ k15. Unit 10.14 Use of Hyaluronan-Derived Hydrogels for Three-Dimensional Cell Culture and Tumor! s+ E3 K' |5 `3 V/ r6 Q: |
Xenografts+ w7 K: c& m1 e( W
16. Unit 10.15 Generation of Micropatterned Substrates Using Micro Photopatterning
; R" x( x# t4 K8 t6 m1 a1 P17. Unit 10.16 Preparation of Hydrogel Substrates with Tunable Mechanical Properties3 [8 j# x# G- N2 n' M: t5 g
18. Unit 10.17 Engineering Three-Dimensional Collagen Matrices to Provide Contact Guidance during 3D
- \7 `' _" B! e8 F# t! O8 NCell Migration: v: f+ a% l, v; q) u% [
19. Unit 10.18 Imaging Cells in Three-Dimensional Collagen Matrix
1 D4 U- F3 M! p: W9 h7 e) O$ S4 T' `13. Chapter 11 In Vitro Reconstitution0 ^% C3 d5 [2 R( e7 ^# B: ~
1. Introduction/ j& W) X$ e1 }
2. Unit 11.1 Overview of Eukaryotic In Vitro Translation and Expression Systems
2 s; x* Q- u4 ~. S( ^& q3. Unit 11.2 In Vitro Translation
% d9 V+ I% \3 ?( A) F( y3 Z4. Unit 11.3 In Vitro Analysis of Endoplasmic-Reticulum-to-Golgi Transport in Mammalian Cells; H6 o" J7 Z3 {& y
5. Unit 11.4 Cotranslational Translocation of Proteins into Canine Rough Microsomes$ w3 i. _7 y& r2 V7 z
6. Unit 11.5 In Vitro Analysis of SV40 DNA Replication
' i- y- V7 b! v1 ]% T5 q4 y/ Q7. Unit 11.6 In Vitro Transcription: s1 D6 \! _+ v9 n6 v0 ^1 |' w0 z
8. Unit 11.7 Nuclear Import in Digitonin-Permeabilized Cells
9 [2 k! G5 @% b9. Unit 11.8 In Vitro Translation Using HeLa Extract
0 C6 X3 M% r1 G% w/ h6 d% p" i10. Unit 11.9 Analysis of Eukaryotic Translation in Purified and Semipurified Systems
. E, ?# a5 o0 k2 k11. Unit 11.10 Preparation and Use of Interphase Xenopus Egg Extracts
8 J& d4 K$ K" R+ V; N& J12. Unit 11.11 Analysis of the Cell Cycle Using Xenopus Egg Extracts
6 A% w+ w, j6 F2 H13. Unit 11.12 Analysis of Apoptosis Using Xenopus Egg Extracts4 [% ~, E, X, Y7 o- B; M
14. Unit 11.13 Mitotic Spindle Assembly In Vitro7 G; M: \4 \; E" a0 k9 n
15. Unit 11.14 Analysis of RNA Export Using Xenopus Oocytes" f6 m- J- q1 j, o
16. Unit 11.15 In Vitro Analysis of Peroxisomal Protein Import
- b( P# ?6 z8 n17. Unit 11.16 In Vitro Analysis of Chloroplast Protein Import0 f  S2 z4 `- @, e) W: B0 n5 K" P/ l% @
18. Unit 11.17 In Vitro RNA Splicing in Mammalian Cell Extracts
4 ~: u: L' B3 m0 @6 I: y19. Unit 11.18 Endocytosis Assays in Intact and Permeabilized Cells' l: {  ^; H6 V0 f1 U
20. Unit 11.19 In Vitro Analysis of Yeast Mitochondrial Protein Import
1 _) b! \, ~9 b7 y7 P14. Chapter 12 Cell Motility( E1 \1 P1 _6 f2 f$ ^
1. Introduction
& n, X- c: {+ D0 W2. Unit 12.1 Chemotaxis Assays for Eukaryotic Cells
/ S% v, _+ K/ ?4 O6 C- d. e4 N3. Unit 12.2 Invasion Assays# H. L( \( `6 f; T9 c& ^" O5 H
4. Unit 12.3 Cell Traction
8 u- _( q3 m0 L6 H5 ~5. Unit 12.4 Cell Wound Assays8 P( m) u, B+ M4 Y
6. Unit 12.5 Dictyostelium Cell Dynamics
# l& c( i$ b: q3 _" w+ [1 I, J$ h7. Unit 12.6 Optical Microscopy.Based Migration Assay for Human Neutrophils7 O, L: z; d8 U9 @
8. Unit 12.7 Actin-Based Motility Assay
7 u# d7 w* G' k9. Unit 12.8 In Vivo Marking of Single Cells in Chick Embryos Using Photoactivation of GFP( F& S) F) r( m  }, R( P0 g3 J
15. Chapter 13 Organelle Motility
: Q7 r1 |0 O/ I$ O0 s1. Introduction8 c$ @- q/ l+ t7 J2 }; b- x
2. Unit 13.1 Microtubule/Organelle Motility Assays' V8 K% z+ {9 d
3. Unit 13.2 In Vitro Motility Assay to Study Translocation of Actin by Myosin
: N& |, X# C0 Q3 Z6 K4. Unit 13.3 Organelle Motility in Plant Cells: Imaging Golgi and ER Dynamics with GFP5 l- V6 J' r# L" X0 H, N+ `4 o; ^' V
5. Unit 13.4 Movement of Nuclei
2 M* T6 S1 V, k+ W8 E% K0 z! S6. Unit 13.5 Measuring Dynamics of Nuclear Proteins by Photobleaching% E0 Z# U; k- q) P! y. t: b
7. Unit 13.6 Functional Characterization of Proteins Regulating Actin Assembly  ^  j8 W. M# V6 X# N+ L
16. Chapter 14 Signal Transduction: Protein Phosphorylation& r6 L; p0 L, O+ _4 a0 E
1. Introduction
0 ?% E2 o5 N( }+ [: x2. Unit 14.1 Overview of Protein Phosphorylation. b0 c& i8 W% `$ F5 w
3. Unit 14.2 Immunological Detection of Phosphorylation
# J# p- W% c9 ~4. Unit 14.3 The Detection of MAPK Signaling
0 o9 j: k2 e/ H0 G5. Unit 14.4 Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation7 R8 e4 f1 M$ p- R7 d# S
6. Unit 14.5 Phosphoamino Acid Analysis0 J1 [/ J& D4 b; b) z0 D7 J/ t
7. Unit 14.6 Determination of Akt/PKB Signaling' F1 @$ q% }" M/ p' X2 ]
8. Unit 14.7 Analyzing FAK and Pyk2 in Early Integrin Signaling Events* e# a( {  N7 @& l$ S8 X# e! ?
9. Unit 14.8 Rho GTPase Activation Assays
6 f, _0 c, l9 @- A+ S0 x10. Unit 14.9 In Vitro GEF and GAP Assays
, n/ d6 A; w4 z1 c9 f# B11. Unit 14.10 In Vivo Imaging of Signal Transduction Cascades with Probes Based on Forster Resonance. g: U5 Q6 h% I6 N
Energy Transfer (FRET)) ^) A0 z/ \  t5 |5 ^; _9 L+ X
12. Unit 14.11 Biosensors for Characterizing the Dynamics of Rho Family GTPases in Living Cells
) Q4 W) v; X5 B( ~13. Unit 14.12 Analysis of Arf GTP-Binding Protein Function in Cells
, n: I1 n  x9 U# X& P4 A& u' P* G17. Chapter 15 Protein Trafficking
) J! V  j4 d; n  c1. Introduction: {* G0 N1 ^* |) `
2. Unit 15.1 Overview of Protein Trafficking in the Secretory and Endocytic Pathways( P. C% z, H7 o) Q1 V8 j
3. Unit 15.2 Use of Glycosidases to Study Protein Trafficking
4 E1 j2 i* j; l' k: U3 B% Y4. Unit 15.3 Endocytosis: Biochemical Analyses8 @1 E: h* w2 H. ~& ]
5. Unit 15.4 Determining Protein Transport to the Plasma Membrane
" T: {2 D, b2 B- y& S- L6. Unit 15.5 Analysis of Membrane Traffic in Polarized Epithelial Cells$ D. |, R7 Y/ ?/ H& S0 r$ F( h
7. Unit 15.6 Analysis of Protein Folding and Oxidation in the Endoplasmic Reticulum6 g! _* E) u* M6 s
8. Unit 15.7 Measurements of Phagocytosis and Phagosomal Maturation4 ]; F9 I& Y( ]6 D3 ]! \, x, D, o
9. Unit 15.8 Analysis of Protein Transport to Lysosomes
: R* n7 l4 H! B+ z/ E' ]5 `10. Unit 15.9 Studies of the Ubiquitin Proteasome System
5 a- r) j* u8 W$ g; Y" p; Y11. Unit 15.10 Measuring Retrograde Transport to the Trans-Golgi Network
& I" w: r$ p3 Q8 n2 z$ i. `% \% U12. Unit 15.11 Assays for Regulated Exocytosis of Mast Cell Granules% f3 ], u# _4 `
13. Unit 15.12 Analysis of Regulated Secretion Using PC12 Cells
  b* N/ K/ a4 t. J0 r14. Unit 15.13 Analysis of Endocytic Trafficking by Single-Cell Fluorescence Ratio Imaging
2 E! l9 i) @8 D# ]15. Unit 15.14 Quantitative Analysis of Endocytosis and Turnover of Epidermal Growth Factor (EGF) and! y% Z: G" s# q/ C1 ~
EGF Receptor
7 u% `0 U" H0 O1 o; `! `( o16. Unit 15.15 Documenting GLUT4 Exocytosis and Endocytosis in Muscle Cell Monolayers
1 A% c& m" Y1 Z- i( f+ H/ ^18. Chapter 16 Antibodies as Cell Biological Tools, g3 B' A9 `' b7 P4 g0 E5 w
1. Introduction3 g  k3 r( A8 u0 t/ d& R
2. Unit 16.1 Production of Monoclonal Antibodies
. I4 J! k* c2 F0 u$ M, z$ _3. Unit 16.2 Production of Polyclonal Antisera% A' ?2 Z+ J. j' D' \& F) D
4. Unit 16.3 Purification of Immunoglobulin G' o+ c2 r# f) O/ q# w' b+ }
5. Unit 16.4 Fragmentation of Immunoglobulin G
6 Q6 d- x0 I2 l! l5 p* E! y6. Unit 16.5 Antibody Conjugates for Cell Biology2 g' Z, b7 ]) ^, R3 G
7. Unit 16.6 Production of Antibodies That Recognize Specific Tyrosine-Phosphorylated Peptides
8 Z2 N- o8 _8 o, x' W19. Chapter 17 Macromolecular Interactions in Cells
1 @0 ~9 K9 r$ f4 }0 \, I# j  ~1. Introduction
. R2 K- X% R) A, i7 X( E3 U2. Unit 17.1 Imaging Protein-Protein Interactions by Fluorescence Resonance Energy Transfer (FRET)1 c) t+ f& }: P# n: G5 \- P
Microscopy5 ]/ a' m4 s$ }& e/ |7 ]
3. Unit 17.2 Identification of Protein Interactions by Far Western Analysis
* L( @( p# c2 g6 f% l* U# o" N7 j4. Unit 17.3 Interaction Trap/Two-Hybrid System to Identify Interacting Proteins
( h7 e5 S2 N5 x3 P0 e7 }5. Unit 17.4 Mapping Protein-Protein Interactions with Phage-Displayed Combinatorial Peptide Libraries2 ~' m8 p/ V0 U" v
6. Unit 17.5 Protein-Protein Interactions Identified by Pull-Down Experiments and Mass Spectrometry6 O. e7 t6 {5 {2 f/ P9 `! v( ^
7. Unit 17.6 Measuring Protein Interactions by Optical Biosensors1 _, V0 D  G) W* L& [7 C
8. Unit 17.7 Chromatin Immunoprecipitation for Determining the Association of Proteins with Specific' g. E; R8 q1 A& q7 x- t5 T7 j
Genomic Sequences In Vivo
# g: Q6 @+ J6 x: p; b9. Unit 17.8 Isothermal Titration Calorimetry
5 x7 d5 m) a9 ]- W10. Unit 17.9 Rational Design and Evaluation of FRET Experiments to Measure Protein Proximities in Cells
$ I, ]8 J( K& v( N11. Unit 17.10 Identification and Analysis of Multiprotein Complexes Through Chemical Crosslinking2 M3 f# t2 j7 L. b& g0 F
12. Unit 17.11 Visualization of RNA Using Fluorescence Complementation Triggered by Aptamer-Protein
" _; E6 \0 w- d: A/ l; q/ j5 ]+ nInteractions (RFAP) in Live Bacterial Cells
7 Z3 l  r% q; r$ F- ^20. Chapter 18 Cellular Aging and Death/ i' k6 x" b/ }! B; K
1. Introduction
' K* d9 ]5 Y0 u7 a- e/ v- }" z2. Unit 18.1 Current Concepts in Cell Death& L) ]. `, L. X+ v% s8 x3 _7 y
3. Unit 18.2 Analysis of Caspase Activation During Apoptosis( o) F  `2 o4 l' ]8 _, t! g
4. Unit 18.3 Assessment of Apoptosis and Necrosis by DNA Fragmentation and Morphological Criteria
: i" w/ W5 O" }5. Unit 18.4 Quantitative Fluorescence In Situ Hybridization (Q-FISH)
3 ^/ T2 U2 g1 i! A7 L6. Unit 18.5 Analysis of Mitochondrial Dysfunction During Cell Death
: q4 X( D# H7 q7. Unit 18.6 Analysis of Telomeres and Telomerase; f$ ?9 }3 j0 Y; y
8. Unit 18.7 Nonisotopic Methods for Determination of Poly(ADP-Ribose) Levels and Detection of8 c2 e' e2 }' e) N  E) D) l. W& I
Poly(ADP-Ribose) Polymerase0 ~9 v' k4 J3 d% q+ w. l
9. Unit 18.8 Flow Cytometry of Apoptosis
  T  @9 Z; O2 c. Q10. Unit 18.9 Analysis of Cellular Senescence in Culture In Vivo: The Senescence-Associated -Galactosidase' b: j6 P8 H% v  \8 p$ B& B  b
Assay
) H) ]6 x  I; p4 F! _6 t: r11. Unit 18.10 High-Throughput Live Cell Imaging of Apoptosis
3 k- \5 ?2 \: L0 k8 A$ v21. Chapter 19 Whole Organism and Tissue Analysis
/ P- Q8 D! n$ O0 `7 ~: m) y/ G1. Introduction& w$ k7 i& x2 w$ N$ [3 K7 f
2. Unit 19.1 Overview of Metastasis Assays
7 A8 p0 t8 f! r% S3 I3. Unit 19.2 Tail Vein Assay of Cancer Metastasis! `# j. h: b/ a! S& s9 P. c* ^
4. Unit 19.3 Microanalysis of Gene Expression in Tissues Using T7-SAGE: Serial Analysis of Gene
, l& ?6 G# Y3 {. O" v- _Expression After High-Fidelity T7-Based RNA Amplification
& g1 v2 e# m6 C5. Unit 19.4 SAGE Analysis from 1 兪g of Total RNA  m" S1 X* [. c6 i5 m
6. Unit 19.5 The Chick Chorioallantoic Membrane as an In Vivo Angiogenesis Model7 ~5 S; @+ e, k2 j% B# F2 z* m
7. Unit 19.6 Experimental Metastasis Assays in the Chick Embryo% T0 m" F  ~% V
8. Unit 19.7 Imaging Tumor Cell Movement In Vivo& s" Y* J8 X2 c( K$ e& U' r9 H
9. Unit 19.8 Embryonic Organ Culture
& w. t- c8 f8 T$ ~; [) `10. Unit 19.9 Three-Dimensional Tissue Models of Normal and Diseased Skin: S& o5 ]" f/ A' Q+ C; z7 g
11. Unit 19.10 Overview: Engineering Transgenic Constructs and Mice
) q1 B9 b3 P# Z" O0 W12. Unit 19.11 Generation of Transgenic Mice2 C' R+ I. C! ?3 x- |
13. Unit 19.12 Overview: Generation of Gene Knockout Mice& F- ?0 W! r: T( s  V$ ^
14. Unit 19.13 Manipulation of Mouse Embryonic Stem Cells for Knockout Mouse Production
7 ]4 l: K/ ~2 J9 s% g- {( Q15. Unit 19.14 Generation of Gene Knockout Mice by ES Cell Microinjection# J. e0 J: d- D( t/ z
22. Chapter 20 Expression and Introduction of Macromolecules into Cells
, {9 }/ V, M% A  r; h' z7 C1. Introduction, @' t) G8 J7 E
2. Unit 20.1 Direct Introduction of Molecules into Cells6 d) M5 v" C; a8 N
3. Unit 20.2 Protein Transduction: Generation of Full-Length Transducible Proteins Using the TAT System7 L$ O6 H( i7 C4 z$ e
4. Unit 20.3 Calcium Phosphate Transfection
/ G- `& w; [# W5 N  D5. Unit 20.4 Transfection Using DEAE-Dextran8 `! M, v8 L' y* u& E
6. Unit 20.5 Transfection by Electroporation
" c& ^; p$ m- N. r( ?$ z  c& W; R7. Unit 20.6 Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents$ T  Z. d9 f3 ~
8. Unit 20.7 Optimization of Transfection( f5 M7 Q. r8 j# H! j) q0 p
9. Unit 20.8 Inducible Gene Expression Using an Autoregulatory, Tetracycline-Controlled System  @* C- n! T. m  G& H, v
23. Chapter 21 Fluorescent Protein Technology
# P5 k% z; w, D* d' _, z1. Introduction
8 N) U, Q$ k% t. |. t4 T' \1 T2. Unit 21.1 Measuring Protein Mobility by Photobleaching GFP Chimeras in Living Cells
# @! H' t, H2 j( L5 b3. Unit 21.2 Fluorescence Localization After Photobleaching (FLAP)  x7 c3 ?1 b8 X" o* l
4. Unit 21.3 Visualization of Protein Interactions in Living Cells Using Bimolecular Fluorescence  T* C+ x4 L8 L# y$ ]  V
Complementation (BiFC) Analysis
. f6 x7 ?( x; @' s- U+ Y# ^5. Unit 21.4 Design and Use of Fluorescent Fusion Proteins in Cell Biology
7 t3 r& X9 P% ?3 C7 S6. Unit 21.5 The Fluorescent Protein Color Palette
9 J2 P/ y0 c, L8 r# }8 s1 ^/ q4 L7. Unit 21.6 Photoactivation and Imaging of Photoactivatable Fluorescent Proteins% M( x3 _2 `6 p/ u6 ~! ?
24. Chapter 22 Cell Biology of Chromosomes and Nuclei' G/ w) M3 k. Z9 c* F
1. Introduction* C" d# A) I+ d) B, |1 m8 P
2. Unit 22.1 Overview of Cytogenetic Chromosome Analysis
1 u; I& F+ Z, \  s) n3 C/ k3. Unit 22.2 Preparation of Cytogenetic Specimens from Tissue Samples
8 t( O* F2 ^- l. d* P8 W/ }4. Unit 22.3 Traditional Banding of Chromosomes for Cytogenetic Analysis5 r5 d9 N& Z( D0 W. r# U2 S
5. Unit 22.4 Fluorescence In Situ Hybridization (FISH)
% G3 i4 q; q; n6. Unit 22.5 Multi-Color FISH Techniques
' x$ P  C( R: h2 ?' `9 y' a7. Unit 22.6 Comparative Genomic Hybridization- N! J$ N( f  F5 u
8. Unit 22.7 Sister Chromatid Exchange; M, I8 l# S! J
9. Unit 22.8 Detection of Mitotic Figures and Components of the Mitotic Machinery
& y% w' a& P. |7 R, q10. Unit 22.9 Assembly and Micromanipulation of Xenopus In Vitro.Assembled Mitotic Chromosomes
# y9 J" ], |7 @4 T+ x2 G  ^11. Unit 22.10 Replication Labeling with Halogenated Thymidine Analogs5 G1 n+ W$ S* n1 b% c, u6 [
12. Unit 22.11 Assays for Ribosomal RNA Processing and Ribosome Assembly
4 f5 |. f- l/ p8 [" n13. Unit 22.12 Visualization and Measurement of DNA Methyltransferase Activity in Living Cells6 G5 q: k' z: H7 X, W% O0 c. U; o4 m
14. Unit 22.13 Monitoring mRNA Export
7 S2 n0 ^/ B6 `9 D; g3 l3 U" W* b15. Unit 22.14 Analysis of DNA Replication in Saccharomyces cerevisiae by Two-Dimensional and Pulsed-; N. G6 R, \4 W; F: [$ ~* r
Field Gel Electrophoresis
/ p, J3 o6 b1 O, I$ G" W7 v. U* C25. Chapter 23 Stem Cells% K8 S# Y# S- J. @
1. Introduction
+ N* v" c8 k6 ^7 z7 F2. Unit 23.1 Stem Cells: An Overview& Z6 p. e* u6 w4 t( V
3. Unit 23.2 Mouse Embryonic Stem Cell Derivation, and Mouse and Human Embryonic Stem Cell Culture
( `" \" H  Q9 d5 M5 N  o6 u' e# H- oand Differentiation as Embryoid Bodies0 T% q: d* Y8 q4 m; R' R5 G2 n9 c
4. Unit 23.3 Maintenance and In Vitro Differentiation of Mouse Embryonic Stem Cells to Form Blood+ p. j8 _/ r; q. ]6 X9 h) z# ~0 J" e
Vessels) p4 q" n; w$ e
5. Unit 23.4 Differentiation of Mouse Embryonic Stem Cells and of Human Adult Stem Cells into
4 o9 l4 i# |4 U6 k( V# s+ N6 CAdipocytes# f) {# D) @# F% K. _
6. Unit 23.5 Induction of ES Cell.Derived Cartilage Formation
7 {' e  F8 I) t  }" l7. Unit 23.6 Hematoendothelial Differentiation of Human Embryonic Stem Cells5 A! d( r) y! V9 E! \' R; p. N
8. Unit 23.7 Neural Differentiation of Human ES Cells6 u  ~9 z2 H# `
26. Chapter 24 Lipids% t' K: i: e5 W: T; w- V
1. Introduction
+ ?# t. G# U1 ?6 A" `+ c5 T) q2. Unit 24.1 Using Fluorescent Sphingolipid Analogs to Study Intracellular Lipid Trafficking$ c" _+ h+ Z( I0 r0 p
3. Unit 24.2 Fluorescent Detection of Lipid Droplets and Associated Proteins, G% O+ B1 y4 t* S! X
4. Unit 24.3 Making Giant Unilamellar Vesicles via Hydration of a Lipid Film
4 z2 y/ n' D7 @5 s+ M& c5. Unit 24.4 Visualization of Cellular Phosphoinositide Pools with GFP-Fused Protein-Domains
0 c% q. E; h" v& W27. Chapter 25 Nanotechnology  Z8 d1 q, z$ X, }  A2 ~
1. Introduction7 I9 A5 Y# ~. q
2. Unit 25.1 In Vivo Imaging Using Quantum Dot.Conjugated Probes
7 X& T4 f2 |+ H, v5 b+ y% ?; S3. Unit 25.2 Fabrication and Application of Nanofibrous Scaffolds in Tissue Engineering8 Z" K# }8 @) }" w$ S# H! }
28. Chapter 26 Viruses; D& k8 V( i8 ]# ^: k* H
1. Introduction4 v2 A" V: B8 x3 P
2. Unit 26.1 Production of Papillomavirus-Based Gene Transfer Vectors
# N$ k" E2 d/ h. ^' t- q9 j3. Unit 26.2 BK Virus (BKV): Infection, Propagation, Quantitation, Purification, Labeling, and Analysis of1 V3 m  T0 |8 L- A
Cell Entry
9 E4 ^7 W6 n5 y+ s6 ^( S4. Unit 26.3 Methods Used to Study Respiratory Virus Infection
- x" M" _/ |- }( Y1 X5. Unit 26.4 Compartmented Neuron Cultures for Directional Infection by Alpha Herpesviruses
0 C$ Q$ B) Y3 i3 J. S% a  M7 `6. Unit 26.5 HIV-1 Interactions with Cells: From Viral Binding to Cell-Cell Transmission8 ]) m9 y) g/ i3 D
29. Chapter 26 Lipids
+ q9 h% {. i) K' q" d1. Unit 26.6 Methods for Monitoring Dynamics of Pulmonary RSV Replication by Viral Culture and by
* @4 ]3 X: w& e8 }  {- F! @Real-Time Reverse Transcription.PCR In Vivo: Detection of Abortive Viral Replication
+ f$ \; ~" x6 h; h# s; k. R+ S30. Chapter 27 RNA-Based Methods in Cell Biology
  N6 e2 Q6 T; E+ I) x  _& d1. Introduction
7 M5 M; o/ b- o3 H, \2. Unit 27.1 Silencing of Gene Expression in Cultured Cells Using Small Interfering RNAs, @; P7 @/ N1 M9 {
3. Unit 27.2 Gene Down-Regulation with Short Hairpin RNAs and Validation of Specificity by Inducible" i- U/ v. y, L1 _. w8 l+ `; ?
Rescue in Mammalian Cells7 g) g" r4 ^1 l) E* {) k
31. Appendix 1 Useful Information and Data
. e. r* D% k" h3 k1. 1A Useful Measurements and Data& o9 v7 E! b! A
2. 1B Compendium of Drugs Commonly Used in Cell Biology Research4 Q) L- Z' K& K
3. 1C Identification of Motifs in Protein Sequences
* r; l5 ^5 x" q# K+ c7 _2 I$ j4. 1D Safe Use of Radioisotopes
- a& l0 [5 U1 {5. 1E Absorption and Emission Maxima for Common Fluorophores' k  s9 Q! `# g/ n; P% Y% S- ^
6. 1F Importing Biological Materials
& n7 h) n- q7 s" z+ @' z  |1 F7. 1G Centrifuges and Rotors
; Z) s$ l9 J/ O1 x# G8. 1H Internet Basics for Biologists& W$ j3 n: N; D5 F7 U1 N2 I
32. Appendix 2 Laboratory Stock Solutions and Equipment8 J1 j# ]8 q" a  C
1. 2A Common Stock Solutions, Buffers, and Media
, b) ?) Q$ N) V# R  K2. 2B Medium Formulations! X' m) E! G2 I
3. 2C Standard Laboratory Equipment
: E* Z' A, o) D' N& C7 x; g/ M33. Appendix 3 Commonly Used Techniques! L; [6 o2 ?7 r1 Q9 Q& p: u, _4 B
1. 3A Molecular Biology Techniques7 u: N- p2 H+ `" `2 C0 Z
2. 3B Spectrophotometric Determination of Protein Concentration
+ P' P# Q  X+ n1 r, P8 l- F3. 3C Dialysis and Concentration of Protein Solutions, _6 A+ j. J, k" E# ?4 `
4. 3D Quantification of DNA and RNA with Absorption and Fluorescence Spectroscopy# U: p* z+ t! f) V
5. 3E Silanizing Glassware  g5 u% N! |5 j& d, u( B: K# @- C8 u
6. 3F Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization& R7 |% r1 y8 R" y
7. 3G Micro RT-PCR
4 Z2 S0 a) o5 N$ D8. 3H The Colorimetric Detection and Quantitation of Total Protein. w2 [# K" D& p4 m. s* h4 z7 o/ r
34. Appendix Suppliers
* U! ?* Q  i1 Q4 l; N1. Selected Suppliers of Reagents and Equipment0 F" c0 j9 ?( B0 D5 n. `, `

/ Q0 y) V; h) a+ P2 M; U

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